Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics.

Abstract

beta-Adrenergic modulation is one of the most important regulatory mechanisms of ion channel function. Only recently, however, have beta-adrenergic effects on cardiac Na+ channel activity been recognized, and some diversity of effects has been reported in different preparations. We report studies of protein kinase A-dependent phosphorylation effects on cardiac Na+ current using the macropatch on-cell mode voltage-clamp technique to maintain cytoplasmic composition intact. During the first 5 minutes after addition of 8-(4-chlorophenylthio)cAMP to the bath, the midpoints of both voltage-dependent availability and conductance shifted in the hyperpolarizing direction an average of -7.5 +/- 2.8 mV (n = 31). Moreover, these effects were not species specific; similar results were obtained in canine, rabbit, and guinea pig myocytes, and a similar shift occurred after exposure to 5 microM isoproterenol. Maximum conductance did not change, nor did single-channel conductance. The shifts of conductance and voltage-dependent availability that were induced by protein phosphorylation were distinct from and independent of the slow background shift in kinetics. We measured the background shift to be less than 0.3 mV/min and to be restricted to the channels within the patch. Pretreatment of cells with a blocker of protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), prevented the effect of 8-(4-chlorophenylthio)cAMP while not affecting the background shift in kinetics. Although clearly not the result of addition of a negatively charged phosphate to the inside face of the channel, cAMP-dependent phosphorylation affects the voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor.

Effects of PKC and PKA Inhibitors on the cAMP-Stimulant-Induced Enhancement of Tetrodotoxin-Resistant Na+ (Nav1.8) Currents

“…Similarly, we found that application of 0.5 μM Ro-31-8425 application blocked an increase in Nav1.8 currents induced by either 8-Br-cAMP or the cAMP analogue forskolin [23]. This was supported by the fact that application of a PKC inhibitor (staurosporine or PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] ) caused a marked inhibition of the forskolin-induced increase in the G V1/2 base (i.e. the percentage change in G at baseline V 1/2 ) [24,36,46].…”

“…The application of the PKC inhibitor PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] significantly suppresses the forskolin-induced increase in the Nav1.8 current but the PKA inhibitor WIPTIDE has no significant effect on the PKC activator phorbol12, 13-dibutyrate (PDBu)-induced increase in the current [36]. When considering these results together, it is possible that PKCinduced phosphorylation of the channel protein at serine 1506 is required to enable PKA-induced phosphorylation of other sites on the channel protein suggested by Li et al [40] in a relationship between PKC and PKA on the convergent regulation of Na + channels.…”

“…The slow onset of Nav1.9 channel opening probably implies that this type of Na + channels makes a minor contribution to the action potential amplitude [25]. The majority of voltage-gated sodium channels (VGSCs) are inhibited by either protein kinase C (PKC) or protein kinase A (PKA) [26][27][28][29][30][31][32]. The VGSC into the skeletal and cardiac muscles is not significantly affected by the activation of PKA [33,34].…”

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

“…cAMP then activates protein kinase A (PKA) and phosphorylates proteins that contain the PKA-Ser/ Thr motif. PKA-mediated phosphorylation of voltage-gated Na ϩ channel subunit 1.5 (Na v 1.5) channels typically results in increases in both current amplitude and the rate of current decay (10,15,17,21,34). In addition to this classic paradigm, another pathway has been identified through studies in which pipette dialysis was used to apply the PKA inhibitor (PKI) peptide to adult ventricular myocytes (15).…”

L-Type Ca 2+ Currents Overlapping Threshold Na + Currents

“…16,17 In feline myocytes, ISO caused small Ca transients to be seen over the same general voltage range as I Na activation ( Figures 1A and 1D). These Ca transients were abolished by thapsigargin (data not shown): evidence that they represent SR Ca release.…”